Lipid nanocapsules, preparation process and use as medicine

ABSTRACT

The invention concerns nanocapsules, in particular with an average size less than 50 nm, consisting of an essentially lipid core liquid or semiliquid at room temperature, coated with an essentially lipid film solid at room temperature having a thickness of 2-10 nm. The invention also concerns a method for preparing same which consists in producing a reverse phase of an aqueous emulsion brought about by several temperature raising and lowering cycles. Said lipid nanocapsules are particularly designed for producing a medicine.

The present invention relates to lipid nanocapsules, to a process forpreparing them and to their use for manufacturing a medicament intendedespecially to be administered by injection, orally or nasally.

In recent years, many groups have developed the formulation of solidlipid nanoparticles or lipid nanospheres (Müller, R. H. and Mehnert,European Journal of Pharmaceutics and Biopharmaceutics, 41(1): 62-69,1995; W., Gasco, M. R., Pharmaceutical Technology Europe: 52-57,December 1997; EP 605 497). This is an alternative to the use ofliposomes or polymer particles. These lipid particles have the advantageof being formulated in the absence of solvent. They allow theencapsulation of both lipophilic and hydrophilic products in the form ofion pairs, for example (Cavalli, R. et al., S.T.P. Pharma Sciences,2(6): 514-518, 1992; and Cavalli, R. et al., International Journal ofPharmaceutics, 117: 243-246, 1995). These particles may be stable forseveral years in the absence of light, at 8° C. (Freitas, C. and Müller,R. H., Journal of Microencapsulation, 1 (16): 59-71, 1999).

Two techniques are commonly used to prepare lipid nanoparticles:

-   -   homogenization of a hot emulsion (Schwarz, C. et al., Journal of        Controlled Release, 30: 83-96, 1994; Müller, R. H. et al.,        European Journal of Pharmaceutics and Biopharmaceutics, 41(1):        62-69, 1995) or of a cold emulsion (Zur Mühien, A. and Mehnert        W., Pharmazie, 53: 552-555, 1998; EP 605 497), or    -   the quench of a microemulsion in the presence of co-surfactants        such as butanol. The size of the nanoparticles obtained is        generally greater than 100 nm (Cavalli, R. et al., European        Journal of Pharmaceutics and Biopharmaceutics, 43(2): 110-115,        1996; Morel, S. et al., International Journal of Pharmaceutics,        132: 259-261, 1996).

Cavalli et al. (International Journal of Pharmaceutics, 2(6): 514-518,1992; and Pharmazie, 53: 392-396, 1998) describe the use of a nontoxicbile salt, taurodeoxycholate, by injection for the formation ofnanospheres greater than or equal to 55 nm in size.

The present invention relates to nanocapsules rather than nanospheres.The term “nanocapsules” means particles consisting of a core that isliquid or semiliquid at room temperature, coated with a film that issolid at room temperature, as opposed to nanospheres, which are matrixparticles, ie particles whose entire mass is solid. When the nanospherescontain a pharmaceutically active principle, this active principle isfinely dispersed in the solid matrix.

In the context of the present invention, the term “room temperature”means a temperature between 15 and 25° C.

One subject of the present invention is nanocapsules with an averagesize of less than 150 nm, preferably less than 100 nm and morepreferably less than 50 nm.

The nanocapsules each consist of an essentially lipid core that isliquid or semiliquid at room temperature, coated with an essentiallylipid film that is solid at room temperature.

Given their size, the nanocapsules of the invention are colloidal lipidparticles.

The polydispersity index of the nanocapsules of the invention isadvantageously between 5% and 15%.

The thickness of the solid film is advantageously between 2 and 10 nm.It is also about one tenth of the diameter of the particles.

The core of the nanocapsules consists essentially of a fatty substancethat is liquid or semiliquid at room temperature, for example atriglyceride or a fatty acid ester, representing 20% to 60% andpreferably 25% to 50% by weight of the nanocapsules.

The solid film coating the nanocapsules preferably consists essentiallyof a lipophilic surfactant, for example a lecithin whose proportion ofphosphatidylcholine is between 40% and 80%. The solid film may alsocontain a hydrophilic surfactant, for example Solutol® HS 15.

The hydrophilic surfactant contained in the solid film coating thenanocapsules preferably represents between 2% and 10% by weight of thenanocapsules, preferably about 8%.

The triglyceride constituting the core of the nanocapsules is chosenespecially from C₈ to C₁₂ triglycerides, for example capric and caprylicacid triglycerides and mixtures thereof.

The fatty acid ester is chosen from C₈ to C₁₈ fatty acid esters, forexample ethyl palmitate, ethyl oleate, ethyl myristate, isopropylmyristate, octyldodecyl myristate, and mixtures thereof. The fatty acidester is preferably C₈ to C₁₂.

The nanocapsules of the invention are particularly suitable forformulating pharmaceutical active principles. In this case, thelipophilic surfactant may advantageously be solid at 20° C. and liquidat about 37° C.

The amount of lipophilic surfactant contained in the solid film coatingthe nanocapsules is set such that the liquid fatty substance/solidsurfactant compound mass ratio is chosen between 1 and 15, preferablybetween 1.5 and 13 and more preferably between 3 and 8.

A subject of the present invention is also a process for preparing thenanocapsules described above.

The process of the invention is based on the phase inversion of anoil/water emulsion brought about by several cycles of raising andlowering temperature.

The process of the invention consists in

-   -   a)—preparing an oil/water emulsion containing an oily fatty        phase, a nonionic hydrophilic surfactant, a lipophilic        surfactant that is solid at 20° C. and optionally a        pharmaceutically active principle that is soluble or dispersible        in the oily fatty phase, or a pharmaceutically active principle        that is soluble or dispersible in the aqueous phase,        -   bringing about the phase inversion of said oil/water            emulsion by increasing the temperature up to a temperature            T₂ above the phase inversion temperature (PIT) to obtain a            water/oil emulsion, followed by a reduction in the            temperature down to a temperature T₁, T₁<PIT<T₂,        -   carrying out at least one or more temperature cycles around            the phase inversion zone between T₁ and T₂, until a            translucent suspension is observed,    -   b) quenching the oil/water emulsion at a temperature in the        region of T₁, preferably greater than T₁, to obtain stable        nanocapsules.

The nanocapsules obtained according to the process of the invention areadvantageously free of co-surfactants, for instance C₁-C₄ alcohols.

The number of cycles applied to the emulsion depends on the amount ofenergy required to form the nanocapsules.

The phase inversion may be visualized by canceling out the conductivityof the formation when the water/oil emulsion is formed.

The process of the invention comprises two steps.

The first step consists in weighing all the constituents, heating themabove a temperature T₂ with gentle stirring (for example magneticstirring) and then optionally cooling them to a temperature T₁ (T₁<T₂).After a certain number of temperature cycles, a water/oil emulsion isobtained.

The phase inversion between the oil/water emulsion and the water/oilemulsion is reflected by a reduction in the conductivity when thetemperature increases until it is canceled out. The average temperatureof the phase inversion zone corresponds to the phase inversiontemperature (PIT). The organization of the system in the form ofnanocapsules is reflected visually by a change in the appearance of theinitial system, which changes from opaque-white to translucent-white.This change takes place at a temperature below the PIT. This temperatureis generally between 6 and 15° C. below the PIT.

T₁ is a temperature at which the conductivity is at least equal to90-95% of the conductivity measured at 20° C.

T₂ is the temperature at which the conductivity becomes canceled out.

The second step consists of a sudden cooling (or quench) of theoil/water emulsion to a temperature in the region of T₁, preferablyabove T₁, with magnetic stirring, by diluting it between threefold andtenfold using deionized water at 2° C.±1° C. added to the fine emulsion.The particles obtained are kept stirring for 5 minutes.

In one preferred embodiment, the fatty phase is a fatty acidtriglyceride, the solid lipophilic surfactant is a lecithin and thehydrophilic surfactant is Solutol® HS15. Under these conditions, T₁=60°C., T₂=85° C. and the number of cycles is equal to 3.

The liquid substance/solid surfactant compound ratio is chosen between 1and 15, preferably between 1.5 and 13 and more preferably between 3 and8.

The oil/water emulsion advantageously contains 1% to 3% of lipophilicsurfactant, 5% to 15% of hydrophilic surfactant, 5% to 15% of oily fattysubstance and 64% to 89% of water (the percentages are expressed on aweight basis).

The higher the HLB value of the liquid fatty substance, the higher thephase inversion temperature. On the other hand, the HLB value of thefatty substance does not appear to have an influence on the size of thenanocapsules.

Thus, when the size of the triglyceride end groups increases, their HLBvalue decreases and the phase inversion temperature decreases.

The HLB value, or hydrophilic/lipophilic balance, is as defined by C.Larpent in Treatise K.342 of Editions TECHNIQUES DE L'INGENIEUR.

The particle size decreases when the proportion of hydrophilicsurfactant increases and when the proportion of surfactants (hydrophilicand lipophilic) increases. Specifically, the surfactant brings about adecrease in the interface tension and thus a stabilization of thesystem, which promotes the production of small particles.

Moreover, the particle size increases when the proportion of oilincreases.

According to one preferred embodiment, the fatty phase is Labrafac® WL1349, the lipophilic surfactant is Lipoid® S 75-3 and the nonionichydrophilic surfactant is Solutol® HS15. These compounds have thefollowing characteristics:

-   -   Labrafac® lipophile WL 1349 (Gattefossé, Saint-Priest, France).        This is an oil composed of caprylic and capric acid (C₈ and C₁₀)        medium-chain triglycerides. Its density is from 0.930 to 0.960        at 20° C. Its HLB value is about 1.    -   Lipoid® S 75-3 (Lipoid GmbH, Ludwigshafen, Germany). Lipoid® S        75-3 corresponds to soybean lecithin. Soybean lecithin contains        about 69% phosphatidylcholine and 9% phosphatidylethanolamine.        They are thus surfactant compounds. This constituent is the only        constituent that is solid at 37° C. and at room temperature in        the formulation. It is commonly used for the formulation of        injectable particles.    -   Solutol® HS15 (BASF, Ludwigshafen, Germany). This is a        polyethylene glycol-660 2-hydroxystearate. It thus acts as a        nonionic hydrophilic surfactant in the formulation. It may be        used by injection (via the iv route in mice LD₅₀>3.16 g/kg, in        rats 1.0<LD₅₀<1.47 g/kg).

The aqueous phase of the oil/water emulsion may also contain 1% to 4% ofa salt, for instance sodium chloride. Changing the salt concentrationbrings about a shift in the phase inversion zone. The higher the saltconcentration, the lower the phase inversion temperature. Thisphenomenon will be advantageous for encapsulating hydrophobicheat-sensitive active principles. Their incorporation may be performedat a lower temperature.

The nanocapsules of the invention may advantageously contain an activeprinciple and may form part of the composition of a medicament to beadministered by injection, especially intravenous injection, orally ornasally.

When the active principle is sparingly soluble in the oily phase, acosolvent is added, for example N,N-dimethylacetamide.

The nanocapsules of the invention are more particularly suitable for theadministration of the following active principles:

-   -   antiinfectious agents, including antimycotic agents and        antibiotics,    -   anticancer agents,    -   active principles intended for the Central Nervous System, which        must cross the blood-brain barrier, such as antiparkinson agents        and more generally active principles for treating        neurodegenerative diseases.

The pharmaceutically active principle may be firstly soluble ordispersible in an oily fatty phase, and in this case it will beincorporated in the core of the nanocapsule. To do this, it isincorporated at the stage of the first step of preparing the oil/wateremulsion which also contains the oily fatty phase, a nonionichydrophilic surfactant and a lipophilic surfactant that is solid at 20°C.

The pharmaceutically active principle may also be of water-solublenature or dispersible in an aqueous phase, and in such a case it will bebound to the surface of the nanocapsules only after the final phase ofpreparing the stable nanocapsules. Such a water-soluble active principlemay be of any nature, including proteins, peptides, oligonucleotides andDNA plasmids. Such an active principle is attached to the surface of thenanocapsules by introducing said active principle into the solution inwhich are dispersed stable nanocapsules obtained after the processaccording to the invention. The presence of a nonionic hydrophilicsurfactant promotes the interaction bonds between the water-solubleactive principle and the free surface of the nanocapsules.

The water-soluble active principle may also be introduced into theaqueous phase during the first step of initial oil/water preparation.

The invention is illustrated by the examples that follow, with referenceto FIGS. 1 to 4.

FIG. 1 is a photograph of the nanocapsules of the invention obtained inExample 1. The scale is 1 cm to 50 nm.

FIG. 2 shows the change in the average particle size as a function ofthe proportion of hydrophilic surfactant (Solutol®).

FIG. 3 shows the change in conductivity as a function of the temperaturefor various salt concentrations. In curve 1, the salt concentration is2.0% by weight. In curve 2, the concentration is 3.4% by weight.

FIG. 4 shows the change in the conductivity of an oil/water (O/W)emulsion described in Example 1, as a function of the temperature afterthree cycles of raising and lowering the temperature between 60 and 85°C.

EXAMPLE 1 Nanocapsules not Containing Active Principle A) Preparation ofthe Nanocapsules

5 g of an emulsion containing 75 mg of Lipoid® S75-3, 504 mg ofLabrafac® WL 1349 lipophile, 504 mg of Solutol® HS15, 3.829 g of waterand 88 mg of sodium chloride are prepared.

The ingredients are combined in the same beaker and placed undermagnetic stirring. Heat is applied until a temperature of 85° C. isreached. The system is allowed to cool to a temperature of 60° C. withmagnetic stirring. This cycle (between 85° C. and 60° C.) is performeduntil a canceling out of the conductivity as a function of thetemperature is observed (FIG. 4). The phase inversion takes place afterthree cycles. At the final cooling, quenching is carried out by adding12.5 ml of distilled water at 2° C.±1° C. to the mixture at 70° C. Thesystem is then maintained under magnetic stirring for 5 minutes.

The particles obtained under the conditions described above, after threetemperature cycles, have a mean size of 43±7 nm. Their sizepolydispersity is 0.071. Transmission electron microscopy usingphosphotungstic acid made it possible to reveal particles with a meansize of about 50 nm (see FIG. 1). Moreover, an observation made byatomic force microscopy in contact mode (Park Scientific Instrumentsapparatus, Geneva, Switzerland) shows that the nanocapsules are indeedsolid at a temperature of 25° C.

B) Change in the Proportions of Hydrophilic Surfactant

Table 1 below shows different formulations of nanocapsules prepared withvariable concentrations of hydrophilic surfactant.

TABLE I Mass % Lipoid 1.51 1.51 1.51 1.51 1.51 1.51 1.51 1.51 S75-3Labrafac ® 10.08 10.08 10.08 10.08 10.08 10.08 10.08 10.08 WL 1349Solutol ® 5.00 7.50 10.08 15.00 20.00 22.50 25.00 30.00 HS 15 Water81.65 79.10 76.60 71.68 66.68 64.18 61.68 56.68 NaCl 1.76 1.76 1.76 1.761.76 1.76 1.76 1.76

Decreasing the concentration of Solutol® HS15 results in an increase inthe mean particle size (FIG. 2). Mean sizes going from 23 to 128 nm arethus observed for Solutol® proportions going from 30% to 5% of the totalformulation, respectively. The size thus depends on the concentration ofhydrophilic surfactant.

C) Changes in the Proportions of Lipoid® and Solutol® Surfactants

Table II below shows formulations of nanocapsules prepared with varioussurfactant concentrations.

TABLE II Mass % A B C Lipoid ® S75-3 0.78% 1.51% 2.35% Labrafac ® WL1349 10.08% 10.08% 10.08% Solutol ® HS 15 5.22% 10.08% 15.65% Water82.16% 76.60% 10.16% NaCl 1.76% 1.76% 1.76% Proportion of 6.00% 11.59%18.00% surfactants

Increasing the proportion of surfactants in the formulation brings abouta reduction in the mean size. Specifically, formulation A givesparticles with a mean size of 85±7 nm (P=0.124). For formulations B andC, the mean sizes become 43±7 nm (P=0.071) and 29±8 nm (P=0.148),respectively.

D) Change in the NaCl Concentration

Table III below shows two formulations of nanocapsules prepared with twodifferent concentrations of NaCl salt.

TABLE III Mass % Lipoid ® S75-3 1.73% 1.70% Labrafac ® WL 1349 5.76%2.84% Solutol ® HS15 2.88% 5.68% Water 87.61% 86.36% NaCl 2.02% 3.40%

Changing the salt concentration brings about a shift in the phaseinversion zone. The higher the salt concentration, the lower the phaseinversion temperature (FIG. 3). This phenomenon will be advantageous forthe encapsulation of hydrophobic heat-sensitive active principles. Theirincorporation may be performed at a lower temperature.

With these formulations, particles similar in size to the previous sizesmay be obtained, despite the different salt concentrations.

EXAMPLE 2 Encapsulation of a Lipophilic Active Principle, Sudan III

The formulation corresponds to that of Example 1: 5 g of the initialemulsion are prepared by weighing out 75 mg of Lipoid® 375-3, 504 mg ofLabrafac® lipophile and 504 mg of Solutol®, 3.829 g of water and 88 mgof sodium chloride. 200 mg of Sudan III dissolved in liquid petroleumjelly are added. The mixture is weighed out in the same beaker andplaced under magnetic stirring. Heat is applied until a temperature of85° C. is reached. The system is allowed to cool to a temperature of 60°C. with magnetic stirring. This cycle (between 85° C. and 60° C.) isperformed three times. At the final cooling, an quenching at 70° C. iscarried out by adding 12.5 ml of distilled water at 2° C.±1° C. Thesystem is then maintained under magnetic stirring for 5 minutes.

The encapsulation of Sudan III made it possible to obtain particles of asimilar size to the particles of Example 1, for the same proportions ofsurfactants and of fatty phase, ie 45±12 nm (P=0.138). To the naked eye,the sample appears a uniform pink.

EXAMPLE 3 Encapsulation of Progesterone

The formulation corresponds to that of Example 1: 5 g of the initialemulsion are prepared by weighing out 75 mg of Lipoid® S75-3, 504 mg ofLabrafac® lipophile and 504 mg of Solutol®, 3.829 g of water and 88 mgof sodium chloride. 10 mg of progesterone are added. The mixture isweighed out in the same beaker and placed under magnetic stirring. Heatis applied until a temperature of 85° C. is reached. The system isallowed to cool to a temperature of 60° C. with magnetic stirring. Thiscycle (between 85° C. and 60° C.) is performed three times. At the finalcooling, an quenching at 70° C. is carried out by adding 12.5 ml ofdistilled water at 2° C.±1° C. The system is then maintained undermagnetic stirring for 5 minutes.

The encapsulation of progesterone makes it possible to obtain particlesof similar sizes to the particles of Example 1, ie 45±12 nm (P=0.112).The progesterone is not found in the aqueous phase at a concentrationabove its solubility. Specifically, a centrifugation at 200 000 rpm for30 minutes gives a light precipitate whose composition was studied byDSC. This precipitate does not contain progesterone. Since progesteroneis virtually insoluble in water, this indicates an incorporation of theactive principle into the nanocapsules.

EXAMPLE 4 Encapsulation of a Busulfan Suspension

A) Suspension of Busulfan (at a Concentration of 0.25 mg/ml)

The first step of the encapsulation of busulfan consists in dissolvingit in N,N-dimethylacetamide. A solution containing 24 mg of busulfan perml of N,N-dimethylacetamide is thus prepared. 175 mg of this solutionare taken and added to 504 mg of Labrafac®. 75 mg of Lipoid® S75-3, 504mg of Solutol®, 3.829 g of water and 88 mg of sodium chloride are alsoweighed out. The initial emulsion is thus at a concentration of 0.88mg/g of emulsion. The ingredients are combined in the same beaker andplaced under magnetic stirring. Heat is applied until a temperature of85° C. is reached. The system is allowed to cool to a temperature of 60°C. with magnetic stirring. This cycle (between 85° C. and 60° C.) isperformed three times. At the final cooling, an quenching at 70° C. iscarried out by adding 12.5 ml of distilled water at 2° C.±1° C. Thesystem is then maintained under magnetic stirring for 5 minutes. Thefinal concentration, ie after quenching, that is to say dilution, is0.25 mg/ml.

The size of the particles obtained is slightly larger than that ofExample 1 on account of the higher proportion of fatty phase (63±5 nm).As for progesterone, busulfan is not found in the aqueous phase at aconcentration above its solubility. Specifically, no crystals arevisible by optical microscopy in the aqueous phase after encapsulation.Now, since busulfan is virtually insoluble in water, this indicates anincorporation of the busulfan into the nanocapsules.

B) Suspension of Busulfan (at a Concentration of 0.50 mg/ml)

A particle suspensation at 0.50 mg/l is prepared under the sameconditions as above after dissolving 50 mg of busulfan in 1 ml ofN,N-dimethylacetamide. 175 mg of this solution are taken and added to504 mg of Labrafac®. 75 mg of Lipoid® S75-3, 504 mg of Solutol®, 3.829 gof water and 88 mg of sodium chloride are also weighed out. The initialemulsion is thus at a concentration of 1.76 mg/ml of emulsion. Theingredients are combined in the same beaker and placed under magneticstirring. Heat is applied until a temperature of 85° C. is reached. Thesystem is allowed to cool to a temperature of 60° C. with magneticstirring. This cycle (between 85° C. and 60° C.) is performed threetimes. At the final cooling, an quenching at 70° C. is carried out byadding 12.5 ml of distilled water at 2° C.±1° C. The system is thenmaintained under magnetic stirring for 5 minutes. The finalconcentration, ie after quenching, that is to say dilution, is 0.50mg/ml.

EXAMPLE 5 Influence of the Nature of the Fatty Substance on the PhaseInversion Temperature

Labrafac®, an oil composed of capric and caprylic acid triglycerides, iscompared with fatty acid esters. It was possible to reveal the influenceof the size of their end groups on the phase inversion temperature. Anincrease in the phase inversion temperature with increasing size of thegroups is observed. Thus, in the myristate series, the change inappearance is visible at 69.5° C. for the ethyl ester, at 71.5° C. forthe isopropyl ester and at 86.5° C. for the octyldodecyl ester. Thisincrease means that an oil-in-water emulsion is more readily obtainedwhen the oil has a lower HLB value (more lipophilic). Specifically, thismore pronounced lipophilic nature brings about an accentuation of thehydrophobic bonds between the surfactant and the oil, and more energy isthus required to invert this system. Moreover, the carbon chain lengthof the fatty acid does not influence the particle size, or the phaseinversion temperature (between C₁₄ and C₁₈). It appears, however, thatthe double bond present in ethyl oleate substantially increases thephase inversion temperature.

The results are given in the table below.

TABLE IV °T change Number of in Particle carbons Double appearance sizeOils (fatty acid) bonds (° C.) (nm) Labrafac ® 8/10 0 77.0 43 ± 7lipophile Ethyl 16 0 69.0 37 ± 15 palmitate Ethyl oleate 18 1 71.5 41 ±5 Ethyl 14 0 69.5 35 ± 13 myristate Isopropyl 14 0 71.5 44 ± 23myristate Octyldodecyl 14 0 86.5 42 ± 16 myristate

The HLB value of the fatty substance does not appear to affect theparticle size significantly.

EXAMPLE 6 Influence of the Nature of the Lipophilic Surfactant on theSize of the Nanocapsules

Various types of lecithin whose phosphatidylcholine proportions rangefrom 40% to 90% were used. The mean particle size increases as thephosphatidylcholine content in the lecithin increases (Table V below).Specifically, for 40% phosphatidylcholine, the size of the nanocapsulesis 35±8 nm, whereas it is, respectively, 43±7 nm and 78±12 nm for aproportion of 75% and 90% phosphatidylcholine in the lecithin. On theother hand, the use of charged molecules did not allow nanocapsules tobe obtained.

TABLE V % of phosphatidyl- Mean particle Type of lipoid choline size(nm) Lipoid ® S45 40 35 ± 8 Lipoid ® S75-3 69 43 ± 7 Lipoid ® S100 90 78± 12 Lipoid ® EPC 98 61 ± 12 Lipoid ® E80 80 72 ± 18

EXAMPLE 7 Lipid Nanocapsules with a Water-Soluble Active PrincipleAttached to their Surface

500 mg of a dispersion of lipid nanocapsules not containing activeprinciple, as described in Example 1, are prepared using the followingformulation:

-   -   Lipoid® S 75-3: 1.51 mass %    -   Labrafac® W1.1349: 10.08 mass %    -   Solutol® HS15: 10.08 mass %    -   Water: 76.6 mass %    -   NaCl: 1.76 mass %

The lipid nanocapsules obtained have a size of 43±7 nm. 50 mg of thedispersion of lipid nanocapsules obtained are diluted in 1 ml of waterand incubated with gentle stirring with an aqueous solution containing50 μg of DNA (pSV β-galactosidase, Promega, France) for one hour in thepresence of a mixture of histones obtained from calf thymus (BoehringerMannheim, Germany). Lipid nanocapsules containing DNA moleculescondensed with the proteins, adsorbed onto their surface, are obtained.

1.-21. (canceled)
 22. Nanocapsules with an average size of less than 150nm, consisting of a core that is liquid or semi-liquid at roomtemperature, and a film coating that is solid at room temperaturecoating said core, wherein: said film consists essentially of a lecithinand contains a PEG-hydroxystearate, said core consists essentially of afatty substance that is liquid or semi-liquid at room temperature andcontains a pharmaceutically active principle.
 23. The lipid nanocapsulesas claimed in claim 22, wherein the average size is less than 100 nm.24. The lipid nanocapsules as claimed in claim 23, wherein the averagesize is less than 50 nm.
 25. The lipid nanocapsules as claimed in claim22, wherein the thickness of the solid film is between 2 and 10 nm. 26.The nanocapsules as claimed in claim 22, wherein the fatty substance isa triglyceride or a fatty acid ester.
 27. The nanocapsules as claimed inclaim 26, wherein the fatty substance represents 20% to 60% by weight ofthe nanocapsules.
 28. The nanocapsules as claimed in claim 27, whereinthe fatty substance represents 25% to 50% by weight of the nanocapsules.29. The lipid nanocapsules as claimed in claim 26, wherein thetriglyceride constituting the core of the nanocapsules is chosen from C₈to C₁₂ triglycerides and mixtures thereof.
 30. The lipid nanocapsules asclaimed in claim 29, wherein the C₈ to C₁₂ triglycerides are capric orcaprylic acid triglycerides.
 31. The lipid nanocapsules as claimed inclaim 28, wherein the fatty acid ester constituting the core of thenanocapsules is chosen from C₈ to C₁₈ fatty acid esters and mixturesthereof.
 32. The lipid nanocapsules as claimed in claim 31, wherein theC₈ to C₁₈ fatty acid esters are chosen from ethyl palmilate, ethyloleate, ethyl myristate, isopropyl myristate and octyldodecyl myristate,and mixtures thereof.
 33. The lipid nanocapsules as claimed in claim 31,wherein the fatty acid ester is C₈ to C₁₂.
 34. The nanocapsules asclaimed in claim 22, wherein the lecithin has a phosphatidylcholineproportion between 40% and 90%.
 35. The nanocapsules as claimed in claim22, wherein the PEG-hydroxystearate represents 2% to 10% by weight ofthe nanocapsules.
 36. The nanocapsules as claimed in claim 22, whereinthe PEG-hydroxystearate is polyethylene glycol-660 2-hydroxystearate.37. The nanocapsules as claimed in claim 22, wherein the lecithin issoybean lecithin.
 38. The nanocapsules as claimed in claim 37,characterized in that the soybean lecithin is Lipoid S75-3®.
 39. Amethod of administering a pharmaceutically active agent, comprisingadministering orally the nanocapsule of claim 22 to a subject in needthereof.
 40. A pharmaceutical composition for oral administration,comprising the nanocapsule of claim 22.